Project Summary/Abstract The process of gene expression requires that the DNA that encodes a gene be transcribed to make a messenger RNA (mRNA) copy, which is then translated to produce a protein. For decades, it was believed that the process of transcription occurred primarily across genes to produce mRNAs. However, over the past few years, with the advent of new technologies, experiments have demonstrated that transcription occurs in a much more widespread fashion. For example, only 1.5% of the human genome encodes proteins, while over 70% is transcribed. While there is little knowledge about the functions of this previously unknown transcription, emerging evidence has pointed towards regulatory roles that are important in human biology and disease. However, many questions remain. This proposal focuses on understanding some of these classes of transcription. The experiments focus around a protein, Spt6, that controls transcription genome-wide. Spt6 is found in organisms from yeast to humans and, in all these organisms it has been shown to play crucial roles. In human cells, Spt6 broadly controls transcription and it has been implicated in cancer. Spt6 interacts with both nucleosomes and RNA polymerase II (RNAPII). Nucleosomes are the main repeating structural units of chromosomes, in which 150 base pairs of DNA are wrapped around an octamer of eight histone proteins. In the absence of Spt6, nucleosomes are not positioned correctly and transcription by RNAPII is greatly altered. This proposal will study Spt6 and transcription in the yeast, Saccharomyces cerevisiae, where transcription is highly conserved with humans. S. cerevisiae is a powerful system as it can be studied by genetic and genome-wide approaches at high resolution. The broad objectives of this proposal are to understand the mechanism by which Spt6 functions, to characterize its role in regulation of transcription across the genome, and to elucidate the roles of two classes of recently identified classes of transcripts that it regulates. Specific Aim 1 will address Spt6 function by studying its interactions with histones by crosslinking purified Spt6 to purified histone octamers and identifying the cross linked amino acids by mass spectrometry. Then, mutations will be created that alter those amino acids in order to study the consequences of impairing the Spt6-histone interaction in living cells. Specific Aim 2 will study intragenic transcription, a poorly understood form of transcription that is normally repressed by Spt6. Experiments will comprehensively identify all intragenic transcripts, determine if they are expressed in response to environmental changes, and study candidates for their possible biological roles. Specific Aim 3 will use a recently developed method, NET-seq, to measure the level of active transcription by Spt6 at high resolution. Spt6 regulation of antisense transcription will be characterized by NET-seq analysis of spt6 mutants, and the regulatory roles of antisense transcription will be studied. As intragenic and antisense transcription, and Spt6 have been associated with human disease, what is learned from these studies will be directly relevant to human health.